Alkyl-substituted vinyl pyridines for mass-coded tagging of proteins

Abstract

Comparative studies of proteins from two or more cell populations are performed by alkylating the cysteine thiol groups of the proteins of all populations with vinyl pyridines in which the pyridine ring in at least one of the populations, in addition to the vinyl substituent, bears one or more alkyl substituents, the various populations thus differing in the alkyl substituents on the pyridine ring. Proteins of one population are thus alkylated with vinyl pyridine itself and those of another population with an alkyl-substituted vinyl pyridine, or both with alkyl-substituted vinyl pyridines using different alkyl groups or a different number of alkyl groups. The mass differential afforded by the difference in alkyl substitution affords a means of pairing individual proteins of one population with those of another while differentiating between the two, using analytical techniques disclosed in the prior art for isotope-coded affinity tagging.

1 . A process for the comparative analyses of each of a plurality of protein populations, said process comprising 
 (a) coupling proteins from each population at cysteine thiol groups to an alkyl-substituted vinyl pyridine of the formula                           in which R is lower alkyl and n is 0 to 4, R and n are uniform for each population of said plurality, and at least one of R and n differs between populations of said plurality, such that the proteins of any one population upon being so coupled are distinguishable from the proteins of all other populations of said plurality by the mass difference caused by the difference in at least one of R and n; and    (b) analyzing said protein populations so coupled while differentiating between individual populations by said mass difference.    
     
     
         2 . The process of  claim 1  wherein n is 1 or 2.  
     
     
         3 . The process of  claim 1  wherein the —CH═CH 2  of said formula is bonded to the carbon atom at either the 2-position or the 4-position of the pyridine ring.  
     
     
         4 . The process of  claim 1  wherein the R group is bonded to the carbon atom at the 2-position, the 4-position, or the 6-position of the pyridine ring.  
     
     
         5 . The process of  claim 1  wherein the alkyl-substituted vinyl pyridine is a member selected from the group consisting of is a member selected from the group consisting of 2-vinyl-4-methylpyridine, 2-vinyl-6-methylpyridine, 4-vinyl-2-methylpyridine, 2-vinyl-4,6-methylpyridine, and 4-vinyl-2,6-methylpyridine.  
     
     
         6 . A kit for the comparative analyses of each of a plurality of protein populations, said kit comprising a plurality of distinct species of alkyl-substituted vinyl pyridines of the formula  
       
         
           
           
               
               
           
         
       
       in which R is lower alkyl and n is 0 to 4, R and n are uniform for each species of said plurality, and at least one of R and n differs between species of said plurality, such that when coupled to a protein at a cysteine thiol group on said protein, the proteins coupled to any one species of said plurality are distinguishable from the proteins coupled to all other species of said plurality by the mass difference caused by the difference in at least one of R and n.  
     
     
         7 . The kit of  claim 6  wherein n is 1 or 2.  
     
     
         8 . The kit of  claim 6  wherein the —CH═CH 2  of said formula is bonded to the carbon atom at either the 2-position or the 4-position of the pyridine ring.  
     
     
         9 . The kit of  claim 6  wherein R group is bonded to the carbon atom at the 2-position, the 4-position, or the 6-position of the pyridine ring.  
     
     
         10 . The kit of  claim 6  wherein the alkyl-substituted vinyl pyridine is a member selected from the group consisting of is a member selected from the group consisting of 2-vinyl-4-methylpyridine, 2-vinyl-6-methylpyridine, 4-vinyl-2-methylpyridine, 2-vinyl-4,6-methylpyridine, and 4-vinyl-2,6-methylpyridine.